Gene expression analysis of laminin-1-induced neurite outgrowth in human mesenchymal stem cells derived from bone marrow.

The mechanisms underlying the differentiation of Mesenchymal stem cells (MSCs) toward neuronal cell type are not clearly understood. Earlier, we reported that laminin-1 induces neurite outgrowth in human MSCs via c-Jun/AP-1 activation through ERK, JNK, and Akt pathways. In this study, we demonstrate that laminin-1 increases the expression of proneural gene, neuroD1 and induces the expression of immediate-early biomarkers of neuronal cell-programming-Egr1, Egr3, PC3, and PC4. Gene expression profiling of MSCs cultured on laminin-1 and Poly-l-lysine for 12 h revealed differential regulation of 267 genes (>1.5 fold, p < 0.05), predominantly in the category of nervous system development and affected the pathways involved in TGF-ß/TNF-α signaling, regulation of MAPK and JNK cascade. Data for 11 selected genes related to nervous system development was validated by real time PCR. Transcriptional regulatory network analysis revealed c-Jun as the key transcription factor regulating majority of differentially expressed genes and identified Disrupted in schizophrenia 1, as a novel target of c-Jun. Modeling and analysis of biological network showed selective induction of Growth Arrest and DNA damage 45 (GADD45B) and repression of NF-κB inhibitor A (NFκBIA). Collectively, our findings provide the basis for understanding the molecular mechanisms associated with laminin-1-induced neurogenic expression in MSCs.

Sodium valproate potentiates staurosporine-induced apoptosis in neuroblastoma cells via Akt/survivin independently of HDAC inhibition.

Sodium valproate (VPA) has been recently identified as a selective class I histone deacetylase (HDAC) inhibitor and explored for its potential as an anti-cancer agent. The anti-cancer properties of VPA are generally attributed to its HDAC inhibitory activity indicating a clear overlap of these two actions, but the underlying mechanisms of its anti-tumor effects are not clearly elucidated. The present study aimed to delineate the molecular mechanism of VPA in potentiating cytotoxic effects of anti-cancer drugs with focus on inhibition of HDAC activity. Using human neuroblastoma cell lines, SK-N-MC, SH-SY5Y, and SK-N-SH, we show that non-toxic dose (2 mM) of VPA enhanced staurosporine (STS)-induced cell death as assessed by MTT assay, PARP cleavage, hypodiploidy, and caspase 3 activity. Mechanistically, the effect of VPA was mediated by down regulation of survivin, an anti-apoptotic protein crucial in resistance to STS-mediated cytotoxicity, through Akt pathway. Knock down of class I HDAC isoforms remarkably inhibited HDAC activity comparable with that of VPA but had no effect on STS-induced apoptosis. Moreover, MS-275, a structurally distinct class I HDAC inhibitor did not affect STS-mediated apoptosis, nor decrease the levels of survivin and Akt. Valpromide (VPM), an amide analog of VPA that does not inhibit HDAC also potentiated cell death in NB cells associated with decreased survivin and Akt levels suggesting that HDAC inhibition might not be crucial for STS-induced apoptosis. The study provides new information on the possible molecular mechanism of VPA in apoptosis that can be explored in combination therapy in cancer.

c-Jun/AP-1 transcription factor regulates laminin-1-induced neurite outgrowth in human bone marrow mesenchymal stem cells: role of multiple signaling pathways.

In this study, we demonstrated that laminin-1 (LM-1) induces neurite outgrowth and enhances the expression of neurofilament-L and MAP2 in human bone marrow mesenchymal stem cells (MSCs). The c-Jun transcription factor was strongly activated by LM-1 during neurite induction. Suppression of c-Jun inhibited the expression of the c-Jun target genes α6 integrin and neurofilament-L, resulting in the loss of neurite outgrowth. Additionally, we found that the LM-1-α6 integrin interaction stimulated phosphorylation of FAK, leading to the activation of JNK and Akt. Pharmacological inhibition of these pathways blocked c-Jun activation and neurite outgrowth. Collectively, our findings suggest that c-Jun/AP-1 activity mediated by JNK, PI3K/Akt and ERK pathways is required for LM-1-induced neurite outgrowth in human bone marrow MSCs.

Laminin-1 induces neurite outgrowth in human mesenchymal stem cells in serum/differentiation factors-free conditions through activation of FAK-MEK/ERK signaling pathways.

Mesenchymal stem cells (MSCs) can be differentiated into cell types derived from all three germ layers by manipulating culture conditions in vitro. A multitude of growth and differentiation factors have been employed for driving MSCs towards a neuronal phenotype. In the present study, we investigated the potential of extracellular matrix (ECM) proteins-fibronectin, collagen-1, collagen-IV, laminin-1, and laminin-10/11, to induce a neuronal phenotype in bone marrow derived human MSCs in the absence of growth factors/differentiating agents. All of the ECM proteins tested were found to support adhesion of MSCs to different extents. However, direct interaction only with laminin-1 triggered sprouting of neurite-like processes. Cells plated on laminin-1 exhibited neurite out growth as early as 3h, and by 24h, the cells developed elaborate neurites with contracted cell bodies and neuronal-like morphology. Function-blocking antibodies directed against alpha6 and beta1 integrin subunits inhibited neurite formation on laminin-1 which confirmed the involvement of integrin alpha6beta1 in neurite outgrowth. Mechanistic studies revealed that cell adhesion to laminin-1 activated focal adhesion kinase (FAK), and mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (MEK/ERK) signaling pathways. Abrogation of FAK phosphorylation by herbimycin-A inhibited neurite formation and also decreased activities of MEK and ERK. Pharmacological inhibitors of MEK (U0126) and ERK (PD98059) also blocked neurite outgrowth in cells plated on laminin-1. Our study demonstrates the involvement of integrin alpha6beta1 and FAK-MEK/ERK signaling pathways in laminin-1-induced neurite outgrowth in MSCs in the absence of serum and differentiation factors.